The lack of improvement in cure rate of many common tumors is amply documented and often ascribed to failure of early detection. Present clinical means for detecting tumor tissue remain in many instances a gross anatomic procedure relying upon various physical findings or radiographic imaging procedures to select a site for histologic sampling. Scintillation imaging techniques with radiopharmaceuticals such as 67Ga-Gallium citrate, 111ln-Bleomycin and 131l-Diiodofluorescein have limited success. These radiolabeled compounds lack specificity and sensitivity, that is, they are not preferentially taken up by tumors. Both 67Ga-Gallium citrate and 111ln-Bleomycin are accumulated in inflammatory or infectious lesions. Currently, all available diagnostic techniques have many drawbacks and limitations in addition to lack of sensitivity and specificity. These include the use of traumatic invasive procedures and the potential for serious complications.
Attempts to “mark” or “tag” tumor cells in order to differentiate them from normal tissues are not new. Various fluorescent compounds such as porphyrins, tetracycline derivatives, acridine orange and toluidine blue or radioactive isotopes have been extensively investigated. With the exception of porphyrin compounds, none of these substances used by earlier investigators are capable of routinely identifying and delineating tumors and tumor margins.
To be effective, an ideal marker substance should: (1) be safe and nontoxic in humans; (2) selectively accumulate only in tumor tissue and not be taken up by normal or inflammatory tissues; (3) be simple to use and involve non-invasive procedures; and (4) be capable of being documented by photographs, radiographs or other recording devices. Unfortunately, the ideal marker or tracer continues to remain elusive.
Technetium-99 m (99mTc) based radiopharmaceuticals have been widely used in the past 15 years. They are by far the safest and the most useful scintigraphic imaging agents developed for Nuclear Medicine procedures. The radionuclide 99mTc has many advantages. It is a pure gamma emitter with a relatively short physical half life of six hours. The gamma photon of 140 KeV energy is compatible with existing conventional scintillation imaging equipment. 99MTc-radiopharmaceuticals can be administered to patients in a much larger dose than many other radiolabeled compounds but produces a minimal radiation health hazard.
For the non-invading nuclear medical diagnosis of tumor, there is ordinarily used gallium citrate (67Ga). While (67Ga) has an accumulating property on tumor cells, it simultaneously possesses the following disadvantages: (1) since its specificity to tumor cells is low and its energy characteristics are not proper, clear and sharp scintigraphy is hardly obtainable; (2) it takes a long time until the radioactivity disappears from the entire body so that many days are needed for the examination; and (3) its half life is 78.1 hours, and the amount of exposure dose against the patient can not be disregarded. For the above reasons, much research has been done to develop an imaging agent having a high specificity to tumor cells to make a quick diagnosis possible.
One of the recent proposals is imaging of tumor cells using a radioisotope-labeled antibody with a high specificity to tumor cell markers. Since the large scale production of a monoclonal antibody by cell culture of hybridoma cells was reported by Milstein et al. (Nature, Vol. 256, p. 495 (1975)), various antibodies specific to tumor-related antigens have been produced, and imaging of tumor cells using these monoclonal antibodies has been extensively tested. The imaging technique using a radioisotope-labeled antibody is generally called a “radioimmunosintigraphy”. Unfortunately, this technique also has inherent problems. For instance, the radioisotope-labeled antibody takes a long time to accumulate on tumor cells and the up-take ratio by these cells is low. Further, the accumulation is done not only by tumor cells, but also by normal organ and tissue cells, and the disappearance of the radioactivity from these organs and tissues takes a long time. For these reasons this technique is has proven impractical.
Studies on the diagnosis of breast cancer have been done with substances specific to steroid hormone receptors such as radioactive iodine-labeled estradiol derivatives (Hanson et al.: American Chemical Society Meeting, Aug. 3-28, 1981, Reference N.U.S.L. 56; Kabalka: Applications of Nuclear and Radiochemistry, Lambrecht, R. M. Morcosn., Eds., Newark, N.J., Pergamon Press, 1981, Chap. 17; JP-A-60-78995). In order to achieve a reliable diagnosis with these receptor-specific substances, the substances are required to satisfy the following conditions: (1) they have to exhibit high affinity and specificity to the receptor; (2) their specific radioactivity must be sufficiently high; and (3) their labeling nuclide must not be liberated in the body. Unfortunately, radioactive receptor-specific substances satisfying all these conditions have not yet been developed.
Various attempts have been made to identify specific tumor sites by simple techniques. For example, it would be desirable to identify the location of tumor cells by localization of a particular tumor marker at the specific tumor site. It would also be desirable to target the specific tumor site with chemotherapeutic agents by introducing substances into the patient's body that are directed to the tumor marker and that deliver a chemotherapeutic agent to the specific tumor site. In spite of such attempts, however, simple delivery systems for targeting tumors in humans do not as yet exist.
Administering a chemotherapeutic agent usually harms many of the normal body cells, often resulting in a worsening of the patient's condition without achieving the desired reduction in tumor size. Historically, this toxicity to normal cells has been a major disadvantage in the treatment of tumors with chemotherapeutic agents. The lack of efficacy of chemotherapy is also attributed to the failure of the freely circulating drug to localize within the tumor cells before it is excreted or taken up by other cells in the body.
Prior attempts to improve treatment of tumors by chemotherapeutic agents includes encapsulation of such chemotherapeutic agents within biodegradable phospholipid micellar particles in the form of vesicles or liposomes. Encapsulation is thought to reduce the toxicity caused by the circulating chemotherapeutic agents. Researchers have also sought to utilize encapsulation to selectively target tumors for delivery of chemotherapeutics agents. Unfortunately, efforts to localize or treat tumors with chemotherapeutic agent-encapsulated targeting particles have not been overly successful.
Localization of tumors such as astrocytomas in the brain in vivo and the determination of the margin between normal tissue and tumor can be useful for surgical, radiotherapeutic and chemotherapeutic approaches to treating the tumor. Although gliomas generally do not metastasize, they do recur locally after surgical resection and carry a grave prognosis. The grave prognosis results in part from the inability to delineate clearly the boundary between tumor and normal brain tissue, and from the restricted permeability of the blood brain barrier to imaging and chemotherapeutic agents.
Monoclonal antibodies prepared against tumors have been proposed for use in the past as effective carrier molecules for the delivery of contrast and radionuclide agents. However, the use of such monoclonal antibodies is accompanied by disadvantages. Antibodies are very large molecules that also can carry cross-reactive antigenic determinants that could cause problems. In addition, the monoclonal antibodies seldom bind more than 70% of cells, even in clonogenic tumors.
In addition to monoclonal antibodies, various synthetic polypeptides, such as polylysine which selectively binds to tumor cells as compared to normal brain cells, have been considered for use as carrier agents for chemotherapeutic agents. Clearly, a need still exists for reliable, safe methods for the imaging, targeting, and treatment of tumors and for substances that can be used in such methods.
In an attempt to satisfy this long felt need, Applicants turned to a family of receptor tyrosine kinases and their cognate ligands that are expressed in the body in particular patterns as candidate molecules for imaging, targeting, and treating tumors. This family, known as the Eph receptor tyrosine kinases, comprise the largest known family of growth factor receptors, and utilize the similarly numerous ephrins as their ligands (Flanagan and Vanderhaeghen, 1998; Gale and Yancopoulos, 1997). The ephrins are unlike ligands for other receptor tyrosine kinases in that they must be membrane-tethered in order to activate their Eph receptors (Davis et al., 1994; Gale and Yancopoulos, 1997). The obligate membrane-attachment of the ephrins provided the first clue that they might act precisely at points of cell-to-cell contact. Based on their means of tethering to the cell membrane, the ephrins can be subdivided into two subclasses. The five members of the ephrin-A subclass (ephrin-A1 to A5) are attached to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor, whereas the three members of the ephrin-B subclass (ephrin-B1 to B3) have a transmembrane region and highly conserved cytoplasmic domains. Ephrin-B ligands primarily interact with the B subset of Eph receptors, consisting of at least six members (Flanagan and Vanderhaeghen, 1998; Gale and Yancopoulos, 1997; Gale and Yancopoulos, 1999). Interactions between ephrin-B ligands and EphB receptors apparently activate bidirectional signaling, in that the cytoplasmic domains of not only the engaged receptor but also of the interacting ligand become phosphorylated on tyrosine residues (Bruckner et al., 1997; Holland et al., 1996).
The ephrins and Ephs were initially studied for their actions in the nervous system, where they seem to play important roles in axonal guidance and in neuronal patterning (Flanagan and Vanderhaeghen, 1998; Gale and Yancopoulos, 1997). More recent studies have begun to focus on roles of these molecules outside of the nervous system. ephrin-B2 and its cognate EphB4 receptor have recently attracted attention in the field of cardiovascular development, based on the vascular defects observed in embryonic mice bearing null mutations in the genes for this ligand and receptor pair (Adams et al., 1999; Gerety et al., 1999; Wang et al, 1998). Normal vascular development initiates with a vasculogenic phase that involves formation of a primitive vascular scaffold, followed by angiogenic stages during which this early vasculature undergoes remodeling and maturation (Risau, 1997). Mouse embryos lacking ephrinB2 and EphB4 suffer fatal defects in early angiogenic remodeling (Adams et al., 1999; Gerety et al., 1999; Wang et al., 1998). Moreover, ephrin-B2 and EphB4 display a remarkably reciprocal pattern of distribution within the developing vasculature—that is, ephrin-B2 marks the endothelium of primordial arterial vessels while EphB4 marks the endothelium of primordial venous vessels (Adams et al., 1999; Gerety etal., 1999; Wang 1998). These distributions suggested that ephrin-B2 and EphB4 are involved developmentally in establishing arterial versus venous identity, perhaps in joining arterioles to venules, and that defects in these processes might account for the early lethality observed in mouse embryos lacking these proteins (Adams et al., 1999; Gale and Yancopoulos, 1999; Gerety et al., 1999; Wang et al., 1998; Yancopoulos et al., 1998).
Despite the remarkably reciprocal distributions of ephrin-B2 and EphB4 during very early vascular development, little is known about the distribution or functions of these proteins as vascular development proceeds, in the quiescent adult vasculature, or when angiogenesis is reinitiated in the adult such as in tumors or in the female reproductive system. To explore these issues, Applicants have exploited a genetically engineered mouse in which the LacZ coding region is used to substitute and report for the ephrin-B2 coding region.